Choleragen has been shown to stimulate adenylate cyclase activity by catalyzing the ADP-ribosylation of a guanine nucleotide-binding regulatory protein (G/F). The effect of this chemical modification on the affinity of the G/F factor for guanine nucleotides was examined to determine the mechanism of choleragen action. Treatment of turkey erythrocyte membranes with choleragen and NAD caused a stimulation in the release of guanine nucleotides from the membranes. Beta-Adrenergic agonists have been shown to stimulate adenylate cyclase by a mechanism in which the catecholamines induce release of GDP from the G/F factor. Guanine nucleotide-binding sites which are affected by the hormone agonists are identical to those which are altered by choleragen treatment, since pretreatment of the membranes with the toxin abolished subsequent stimulation of guanine nucleotide release by catecholamines. Thus, choleragen and hormones may activate adenylkate cyclase by similar mechanisms. Both types of stimulatory agents may act by decreasing the affinity of the G/F for GDP.